The immune systems of mammals have evolved to protect the organism from infectious diseases. Included in the mammalian immune system are many cells that carry out this function by a complex combination of soluble messengers, receptors, adhesion molecules, recognition molecules and signals. Interactions between these components are quite elaborate and take place at specific sites within the organism as well as the primary site of infection. In order to protect the organism from the invasion of viruses, bacteria and other microorganisms, the immune system must be able to distinguish what is self (host) from non-self (invader). The duty is primarily relegated to cell surface molecules known as histocompatibility complex antigens. Each organism has its own set of these molecules to distinguish it from other organisms. In fact, these molecules were discovered when attempts were made to transplant tissues from one host to another.
There are more than thirty of these highly polymorphic molecules that are expressed on the surfaces of virtually all cells. The most important of these molecules are encoded by the major histocompatibility complex (MHC). These genes are categorized as class I, II, or III, depending on their structure, and the role they play in antigen recognition. Class I genes are expressed on virtually all cells, whereas class II gene expression is limited to cells involved in the immune response. It is the class I surface molecules that ‘label’ the cells as foreign or self. The contribution to transplant rejection made by all other histocompatibility genes is unclear.
MHC is defined as a group of genes coding for molecules that provide the context for the recognition of foreign antigens by T lymphocytes. The MHC is mapped on chromosome 6 in humans and on chromosome 17 in mice (see FIG. 1). The class I are A, B, C in humans and K, D, L in mouse and are highly polymorphic. The class II loci (DP, DQ, DR in humans and I-A, I-E in mouse) are also polymorphic (Klein, 1986). The class III genes, encoding components of the complement system, are between the class I and class II genes. Tissue distribution of class I and class II antigens has clear relevance to transplantation. MHC expression is upregulated in donor organs following allotransplantation and such quantitative changes in expression alter the magnitude of the immune response. Under normal conditions the expression of MHC class I genes is developmentally regulated and is modulated by transcriptional and post-transcriptional mechanisms. MHC antigens are hardly detectable until the midsomite stage of embryogenesis (Drezen et al., 1992). In the adult they are expressed on most somatic cells, but with varying levels in different tissues and cell types, even within a given organ (David-Watine et al., 1990). Expression is highest in lymphoid cells, but undetectable in brain cells, sperm cells at certain stages of differentiation, certain cell populations of the placenta and undifferentiated embryonal carcinoma cells (which exhibit a variety of traits characteristic of the early embryo).
Class II gene expression is limited to specialized antigen presenting cells (APC) of the immune system, which include B lymphocytes, macrophages, tissue resident macrophages and dendritic cells of skin and lymph nodes, but can be induced on activated T cells and in other tissues when stimulated by inflammatory cytokines such as interferon. Class I and class II MHC molecules present antigens to T cells to trigger immune responses to various pathogens (Ellis, 1994). In addition, it has been suggested that these molecules play a significant role in macrophage development (Armstrong et al., 1994).
Structurally, MHC molecules are heterodimers. Class I molecules consist of two noncovalently associated subunits: a polymorphic integral membrane heavy chain of approximately 45 kDa encoded in the MHC locus (see FIG. 2) and a smaller subunit called β-2m, a 12 kDa member of the immunoglobulin superfamily. β-2m is a nonpolymorphic product of a non-MHC-complex-linked gene found on chromosome 15 in humans and on chromosome 2 in mice. The function of β-2m is to stabilize the tertiary structure of the heavy chain. The antigen-binding site is represented by a groove formed between the a1 and a2 domains of the heavy chain, which can accommodate peptides of 8 to 10 amino acids in length. For the class I genes, the HLA-A, -B-, and -C loci encode the heavy chain of the class I molecules. For the class II genes, the products of the A genes of each family (DR, DQ, and DP) combine with the products of the B genes of the same family to make the protein αβ heterodimers that are the class II products. The class II genes are referred to by the suffix A or B, encoding a or β chains, respectively. Class II molecules are formed of 2 polymorphic integral membrane proteins, a (33–35 kDa) and β (28–30 kDa) chains (see FIG. 2) that associate by noncovalent interactions. The antigen-binding site is situated between a1 and β1 domains and it can bind longer peptides.
In reality, HLA expression is extremely complex, for example, class I polymorphism is generated by 64 HLA-A, 132 HLA-B, and 39 HLA-C alleles as shown in Table 1 and mainly due to polymorphic residues in the peptide binding pockets. While only two DRA alleles are known, there have been 149 DRB alleles described (see Table 1). Within the DR gene family, some haplotypes express more than one B gene, but only one expressed A gene and a heterozygous individual may inherit two distinct haplotypes. An additional level of complexity is created by the possibility of the expression of haplotype- and isotype-mismatched αβ dimers, i.e., the assembly of an a chain encoded on one haplotype and the b chain from the second haplotype, or the assembly of two chains encoded by the genes of two different class II loci, e.g., DRa Dqβ.
TABLE 1Variability of polymorphic genes inthe MHC of humans (Newell et al., 1996).GeneAlleles1Residues2Variable residues3HLA-A64367110HLA-B132367108HLA-C39367120DRA22291DRB149237691 = number of alleles at that locus2 = number of peptide residues in the mature antigen3 = number of residues that show some variability
In general, only peptides that are derived from autologous proteins and are bound to autologous MHC molecules can be recognized immunologically as ‘self’. All other peptides, by definition, are ‘non-self’, or ‘foreign’. This includes peptides derived from autologous proteins that do not bind to autologous MHC molecules, as well as peptides derived from foreign proteins that are bound to autologous MHC molecules. Further complexity is added by the fact that T cells can effectively engage either autologous or allogeneic MHC molecules. In case of a transplant, APC can be derived either from the host or the graft. There are three ways for T cells to recognize alloantigens: 1) allogeneic peptides bound to autologous MHC class II molecules, 2) allogeneic peptides bound to allogeneic MHC class I molecules, and 3) autologous peptides bound to allogeneic MHC class I or II molecules (self peptides not previously seen). Although these proteins may be present in similar tissues of the graft recipient, the specific peptides selected by the allogeneic MHC molecules have never before been encountered by the recipient's immune system, and technically are considered as alloantigens (VanBuskirk et al., 1994). Acute allograft rejection is mediated by cytotoxic T lymphocytes (CTL) upon recognition of ‘non-self’ antigens bound to MHC class I molecules. T cell activation requires at least two transmembrane signals from T cell surface molecules. One is delivered via the T cell receptor (TcR) complex after productive engagement with MHC class I molecules, another is delivered via at least one of several different adhesion molecules after engagement with their counter-receptor on the tissue cell or APC.
Although the effect of mis-matched MHC can be diminished by immunosuppressive drugs, rejection due to these disparities is still a major barrier to successful organ transplantation. Recombinant DNA technology has made it possible to knock out specific genes in mammals. This technology is predominantly utilized to generate mice lacking in a particular gene. Using these techniques, mice have been created that are missing MHC class I and/or class II antigens. It was hoped that tissues deficient in these molecules would serve as universal donor organs. However, numerous transplantation studies have shown that the absence of these antigens do not make these grafts universally accepted. Furthermore, these MHC-deficient cells cannot perform immune functions to protect the new host from pathogenic invasion.
The goal of transplantation biologists is to successfully replace failing organs or tissues with functional donor organs. However, for transplantation to succeed, two major barriers need to be overcome; first, the availability of suitable donor organs and second, immune rejection. At present, the replacement of failing organs and the treatment of the rejection sequelae is restricted by the limited number of acceptable donors and the need for co-administration of toxic immuno-suppressive drugs in conjunction with long term immuno-suppressive protocols. Current and experimental transplantation protocols rely mainly on sibling donors, other small pools of allogeneic donors, and xenogeneic donors. To overcome these current limitations, there is a growing dependence on tissue matching, non-specific immuno-suppression, and induction of tolerance.
The replacement of the lost function of a diseased organ by transplantation of a healthy organ from a donor to a recipient has been considered a possibility for many years. In practice, this has become clinically feasible only in the last 25 years. Presently, organ transplantation technology is only appropriate in life threatening situations. Two major obstacles have prevented the broad application of transplantation biology. First, the demand for transplantable organs outstrips organ availability, and second, the induction of a vigorous immunologic response results in the rejection of the donor organ (Faustman, 1995). To increase the rate of survival for the transplanted organ, co-administration of toxic immunosuppressive drugs in conjunction with long term immuno-suppressive protocols is common practice. To overcome these limitations, experimental transplantation protocols have been developed.
Although the MHC genes are polymorphic, they are not unique to each individual and it is possible to ‘match’ the tissue donor to the recipient in such a way as to greatly enhance the probability that a graft will be accepted. This is the principle behind the international bone marrow registry. Tissue typing is usually carried out using serological methods (Bollinger and Sanfilippo, 1989), but DNA analysis is used more and more (Bidwell, 1994). Practically speaking, only the HLA can be matched but even then it would be impossible to match all known HLA in an allogeneic transplant situation. The criteria for determining acceptable mismatches depends on several factors such as the particular organ being transplanted and the mechanism of rejection involved. For these reasons transplantation centers place their emphasis on hierarchy of matching different MHC genes, e.g., HLA-DR>-B>-A, and do not rely solely on the number of mismatches (VanBuskirk et al., 1994). One approach to increase the availability of ‘matching’ organs might be to develop a bank of stem cells for each MHC type that could be drawn upon for transplantation, similar to that for bone marrow and cord blood stem cells. The major drawbacks to this approach are: the probability of collecting contaminated stem cells; transference of immune cells along with the graft; the expense of collecting cells, educating prospective donors, and maintaining the infrastructure; ethical issues, and availability donors, as exemplified by the problems associated with obtaining bone marrow cells, grafts, and tissues from minority groups. However, the subject invention does not rely on donor availability or ethnic traits. Contamination will be easily controlled and the infrastructure will be substantially less complex.
It is possible to promote graft acceptance by suppressing the host immune system. This is usually done by administering cyclosporin A, azathioprine, or high doses of steroids, however, these drugs are not without side-effects and act in a non-specific manner. Antibodies directed against the T-cell compartment (anti-CD4 and anti-CD8) or the TCR complex (anti-CD3) have also been used to kill or inactivate the recipient T-cells that maybe responsible for graft damage and rejection (Sell et al., 1996a). These antibodies have also been linked to toxic drugs or natural toxins. The nonspecific nature of this type of approach could conceivably disrupt the fine balance between preventing rejection and the innate ability of the immune system to combat disease.
One of the major goals in the field of transplantation is the achievement of long term, drug-free graft acceptance, preferably associated with donor alloantigen-specific immunologic unresponsiveness. This is the operational definition of allogeneic tolerance (VanBuskirk et al., 1994). It is thought that there are both thymic and extrathymic mechanisms of tolerogenesis that can operate in adult mammals. The presentation of peptides by either the donor or recipient MHC molecules is essential in evoking a T-cell response to transplantation antigens (Lechler and Batchelor, 1982, Benichou, et al., 1997). Both direct and indirect allorecognition are involved in the initial T-cell priming to alloantigens expressed on donor passenger leukocytes within recipient lymphoid organs. It is this T-cell recognition of the donor MHC peptides that is responsible for providing help for cytotoxic T lymphocyte activation and the production of donor-directed antibodies by B lymphocytes. Knowledge about the complexity of regulatory mechanisms (direct and indirect allorecognition) that control T-cell responses to donor MHC determinants during graft rejection can be utilized to design peptide based strategies to block graft rejection. Other strategies have included: 1) the use of bone marrow cells as a vehicle for pretransplant delivery of alloantigens to induce tolerance and long term survival of fully allogeneic allografts (Wong, et al., 1996), 2) the intra-thymic injection of alloantigen (Goss et al., 1993), 3) the in vivo treatment of transplant recipients with antibodies to T lymphocytes (Pearson et al., 1992), or MHC antigens in MHC ‘masking experiments’ (Faustman and Coe, 1991; Faustman, 1995), and to the adhesion molecules such as intercellular adhesion molecule and lymphocyte functional antigen-1 (LFA-1) (Isobe et al., 1992; Faustman, 1995). The induction of tolerance using the monoclonal antibody (BTI-322) in a pre transplant Phase I/II trial as a mechanism to enhance graft survival in renal transplant patients has been shown to reduce graft rejection episodes by 58% compared to conventional triple drug therapy alone.
Other strategies being developed to circumvent transplant rejection and donor shortage include cross-species transplants (xenografts), encapsulation of grafted cells, tissue engineered autologous organs, and the development of genetically engineered ‘universal’ donor cells.
As of 1996, more than 100,000 people in the US, and another 150,000 internationally have benefited from an organ transplant. Despite the 15% annual increase in demand for transplantable organs, the world supply remains static and in some countries is on the decline. One possible solution to alleviate the problem of donor shortage is xenotransplantation, i.e., transplantation of organs from animals to humans. The key issues for successful xenotransplantation are: managing the risk of zoonoses; compatibility of donor organ in size, anatomy, and physiology; overcoming immune rejection of the graft; and the ethical issues (Auchincloss, 1988; Faustman, 1995 and Regalado, 1996). The major problem with animal organs for transplantation goes beyond the compatibility of MHC antigens and introduces another level that deals with the recognition of species-specific antigens. These differences in antigens are responsible for the hyperacute rejection (HAR) phenomenon that can occur within minutes following transplant surgery. A number of studies have centered on introducing the human genes encoding complement inhibitory proteins such as the membrane co-factor protein and decay accelerating factor (CD59) into pigs. Sykes et al., (1991) proposed that the use of xenograft bone marrow transplantation may provide another therapeutic approach to induce hyporeactivity towards a xenogeneic organ donor, while maintaining normal immune function. This approach to tolerize recipients is not feasible for use in human transplantation, primarily due to the high risk associated with myeloablative conditioning regimens required to achieve re-engraftment of allogeneic bone marrow (Sykes et al., 1991).
The science of tissue engineering combines techniques involved in transplantation, cell culture, biomaterials, and genetic engineering. Tissue engineered products include bio-material-based scaffolding for the growth of tissues, implantation of isolated cells, administration of biologically active compounds to effect endogenous tissue and combinations of biomaterials and active compounds. There are a number of studies involving cartilage and bone repair, periodontal repair and peripheral-nerve regeneration, glottic insufficiency, urinary incontinence, post-operative adhesion, metabolic diseases involving liver bioreactors, insulin-dependent diabetes, chronic pain, and neurological diseases and skin repair. The need for autologous tissue will limit the number of centers able to perform these types of services and may make wide-scale use of these therapies impractical.
The primary goal in encapsulation as a cell therapy is to protect allogeneic and xenogeneic cell transplants from destruction by the host immune response. If successful, this approach will eliminate the need for immuno-suppressive drug therapy. Furthermore, the encapsulation will also protect the host from the transplanted cell (potential for dividing cells to cause tumors). Bioencapsulation technologies have shown promise for the encapsulation and transplant of cell populations such as pancreatic islet cells (Siebers et al., 1990; Lanza and Soon-Shiong, 1991) and liver hepatocytes (Chang, 1995; Stange and Mitzner, 1996). This technological approach has been considered for the treatment of hemophilia B, diabetes, chronic pain, and Alzheimer's Disease. The encapsulation of genetically altered cells may offer many advantages over autologous ex vivo gene therapy including their use with ‘universal cells’ containing the desired gene and immunoisolated through encapsulation.
The much touted goal of organ transplantation is to generate ‘generic’ or universally-compatible tissues. In the hunt for this ‘holy grail’ of transplantation, others have attempted to create such a cell by eliminating the expression of class I molecules (U.S. Pat. Nos. 5,574,205; 5,416,260; 5,413,923; and PCT/US90/04178). The development of transgenic mice have made it possible to examine the effect of eliminating class I antigens by creating a ‘knock out’ for the β-2 microglobulin (β-2m) gene. It is well accepted that the function of the β-2m is to stabilize the tertiary structure of the heavy chain of MHC class I and that the absence of the β-2m, from the MHC class I complex adversely affects the transport of the molecule (heavy chain) to the cell surface by the endoplasmic reticulum. This approach to transplantation has not been successful for it appears that cells lacking MHC antigens are targets for natural killer (NK)-mediated cytolysis and are therefore still vulnerable to immune rejection. This argument was further supported by studies demonstrating that rejection could be prevented if NK cells were depleted from the recipient by pretreatment with anti-NK1.1 antibodies. Other published data on NK function indicate that NK cells will kill target cells that have lost or have altered expression of self-MHC antigens: ‘missing self hypothesis’ (Karre et al., 1986 and Carlow et al., 1990). The role of the ‘missing self’ hypothesis in NK-mediated cytolysis is still controversial. Kim et al., (1994) showed that MHC class I surface expression does not influence NK-mediated cytolysis of a target cell and Markmann et al., (1994) found little indication that grafts of non-hematopoietic tissue lacking the expression of MHC class I would be rejected by NK cells.
The theory that tissues devoid of MHC expression could be used as universal donor tissues for transplantation has turned out not to be the panacea for the rejection phenomena. Not only are MHC-deficient cells rejected by the immune system, they are unable to present self and non-self antigens to immune surveillance, seriously undermining the ability of the immune system to recognize the presence of pathogenic organisms. Tissues generated according to the methods of the subject invention have the advantage of being able to overcome immune rejection, both by matching HLA and circumventing NK-mediated cytolysis, without jeopardizing their ability to present antigens in the context of class I and/or class II MHC. No other technology can accomplish all of these things simultaneously.
Two important technologies have recently been developed: the isolation of embryonic stem (ES) cells as permanent in vitro cell lines that can repopulate the blastocyst stage embryo (Evans and Kaufman, 1981) and contribute to the germ-line tissue (Bradley et al., 1984), and the discovery that mammalian cells can recombine introduced vector DNA with a homologous chromosomal target, a process known as gene targeting (Smithies et al., 1985; Thomas and Capecchi, 1987). Gene targeting in ES cells by homologous recombination allows introduction of exogenous DNA sequences into virtually any gene of the germ line so that the gene function can be studied by mutational analysis in vivo. The factors that increase the efficiency of homologous recombination are: a syngeneic background (targeting or homology cassettes derived from the cell to be targeted) (Te Riele et al., 1992), the length of homology between the targeting vector and the genomic DNA of the targeted cell, and the cell's position in the cell cycle (recombination peaks in early S phase) (Capecchi, 1994).
The underlying concept of generating transgenic organisms by homologous recombination is relatively simple: a targeting vector carrying a positive selectable marker flanked by sequences homologous to the genomic target gene is constructed and introduced by transfection into an ES cell line. The homologous flanking sequences enable targeted insertion into the genome and the selectable marker replaces the original wild-type sequence. Subsequently, the successfully targeted ES cell line is injected into blastocysts (3.5 day embryos; 32 cell stage) or co-cultured with morulae (2.5 day embryos; 8–16 cell stage) and contributes to the tissues of the developing animal including the germ line. Breeding will produce homozygous animals exhibiting the phenotype of the inserted mutation in all cells (Galli-Taliadoros et al., 1995).
Although mammalian cells can mediate recombination between homologous DNA sequences very efficiently, they have an even greater predilection for mediating nonhomologous recombination. Recombination occurs via the homologous sequences located within the targeting construct, and does not integrate sequences outside the homology cassette. In contrast, random integration occurs via the ends of the targeting construct, and leads to integration of the entire construct, often in head to tail multimers. Inclusion of a negative selection suicide gene outside the region of homology, therefore allows selection against cells that have undergone non-homologous recombination. Correctly targeted cells will be unaffected by negative selection. The problem is to identify homologous recombination events among the vast pool of nonhomologous recombination events. The invention of methods that lower the background of these nontargeted events and improve screening techniques, namely, positive-negative selection (Mansour et al., 1988), promoterless resistance markers (Schwartzberg et al., 1989), use of polymerase chain reaction (PCR) in screening of pools of clones (Joyner et al., 1989), and polyadenylation (polyA) signal-less markers which produce stable transcripts only if inserted upstream of a genomic polyA signal (Joyner et al., 1989), have made targeted mutations at many nonselectable loci easier to detect.
As can be understood from the above, there remains a need in the art for a means to prepare cells and tissues in which the expression of histocompatibility antigens can be selected for and controlled in order to utilize these cells and tissues in transplantation and other applications.